Browsing by Subject "Velvet"

Multiplex PCR uses several primer pairs, each specific for different DNA sequences in the same reaction, enabling viruses from different families and genera to be recognized by the same test. The new broad-based method of detecting plant viruses, siRNA analysis, is expected to improve the identification of plant viruses as it does not require a prediction of viruses that may be present in the sample. Due to the method, limiting the test to detecting only certain viruses is unnecessary. The aim of this study was to optimize two or more single-phase multiplex RT-PCR tests for the pest laboratory of the Finnish Food Safety Authority (now known as Finnish Food Authority). The tests were for nine published, degenerate primer pairs for the simultaneous identification of Potyvirus, Potexvirus, Tobravirus and Tospovirus genera, Tobamovirus subgroup 1, Nepovirus subgroups a and b, Bromoviridae family and Carmovirus, Dianthovirus, and Tombusvirus, which belong in the Tombusviridae family. Both multiplex RT-PCR tests and siRNA deep sequencing were used to detect plant viruses from infected samples or samples showing viral symptoms. By identifying the viruses in the plant samples, the goal was to estimate the suitability of multiplex RT-PCR tests for identifying multiple RNA viruses from different genera. siRNA analysis was used to ensure the correctness of the multiplex RT-PCR results. In this study, two multiplex RT-PCR tests were created to detect viruses belonging to eight different virus groups. The obtained results serve as a basis for the validation of the multiplex RT-PCR tests. The validation is required to verify the suitability of the method for its intended use and the reliability of the results of the method. The results showed that siRNA deep sequencing was able to detect almost the whole genome from some of the found viruses. Multiplex RT-PCR tests detected Lupine mosaic virus (LuMV) from lupine (Lupinus polyphyllus Lindl.), Arabic mosaic virus (ArMV) from Chinese astilbe (Astilbe chinensis (Maxim.) Franch.) and from quinoa (Chenopodium quinoa Willd.), Tobacco rattle virus (TRV) from moneywort (Lysimachia nummularia L.) and possibly a new Potyvirus species from honey clover (Melilotus albus Medik.). siRNA analysis detected the same viruses, which increases confidence in the optimized multiplex RT-PCR tests.